Comparative gene transfer efficiency of low molecular weight polylysine DNA-condensing peptides

Abstract

In a previous report (M.S. Wadhwa et al . (1997) Bioconjugate Chem. 8, 81–88), we synthesized a panel of polylysine-containing peptides and determined that a minimal repeating lysine chain of 18 residues followed by a tryptophan and an alkylated cysteine residue (AlkCWK 18 ) resulted in the formation of optimal size (78 nm diameter) plasmid DNA condensates that mediated efficient in vitro gene transfer. Shorter polylysine chains produced larger DNA condensates and mediated much lower gene expression while longer lysine chains were equivalent to AlkCWK 18 . Surprisingly, AlkCWK 18 (molecular weight 2672) was a much better gene transfer agent than commercially available low molecular weight polylysine (molecular weight 1000–4000), despite its similar molecular weight. Possible explanations were that the cysteine or tryptophan residue in AlkCWK 18 contributed to the DNA binding and the formation of small condensates or that the homogeneity of AlkCWK 18 relative to low molecular weight polylysine facilitated optimal condensation. To test these hypotheses, the present study prepared AlkCYK 18 and K 20 and used these to form DNA condensates and conduct in vitro gene transfer. The results established that DNA condensates prepared with either AlkCYK 18 or K 20 possessed identical particle size and mediated in vitro gene transfer efficiencies that were indistinguishable from AlkCWK 18 DNA condensates, eliminating the possibility of contributions from cysteine or tryptophan. However, a detailed chromatographic and electrospray mass spectrometry analysis of low molecular weight polylysine revealed it to possess a much lower than anticipated average chain length of dp 6. Thus, the short chain length of low molecular weight polylysine explains its inability to form small DNA condensates and mediate efficient gene transfer relative to AlkCWK 18 DNA condensates. These experiments further emphasize the need to develop homogenous low molecular weight carrier molecules for nonviral gene delivery.